Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene transfer can be achieved in the adult rat heart in vivo by direct injection of plasmid DNA. In this report we define the spatial and temporal limits of reporter gene expression after a single intracardiac injection. pRSVCAT (100 micrograms), in which the Rous sarcoma virus long terminal repeat is fused to the chloramphenicol acetyltransferase reporter gene, and p alpha MHCluc (100 micrograms), in which the alpha-cardiac myosin heavy chain promoter is fused to the firefly luciferase gene, were injected into hearts, and reporter gene activities were assayed at various times. Both chloramphenicol acetyltransferase and luciferase were detectable in 100% of the rats from 1 to 7 days, in 60% of the rats from 17 to 23 days, and in 30% of the rats from 38 to 60 days after injection. Reporter gene activity was largely limited to a 1-2-mm region of the ventricle surrounding the injection site. Closed circular DNA was far more effective than linear DNA in transfecting cells in vivo. The relative strengths of three different promoters, Rous sarcoma virus long terminal repeat, alpha-myosin heavy chain, and alpha 1-antitrypsin, all fused to the luciferase reporter gene were determined. The constitutive viral promoter was approximately 20-fold more active than the cardiac-specific cellular promoter, and the liver-specific cellular promoter was not active at all in the cardiac environment. Thus, direct injection of genes into the heart offers a simple and powerful tool with which to assess the behavior of genes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Behavior of genes directly injected into the rat heart in vivo. 130 14

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.
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PMID:Transcriptional regulatory elements for basal expression of cytochrome P450IIC genes. 132 31

As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.
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PMID:Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer. 137 20

A cultured myocardial cell model was used to examine the role of protein kinase C-dependent pathways in the transcriptional activation of two cardiac muscle genes [myosin light chain 2 (MLC-2) and atrial natriuretic factor (ANF)] during alpha-adrenergic receptor-mediated hypertrophy. Phorbol ester (phorbol 12-myristate 13-acetate) and the alpha-adrenergic agonist phenylephrine both activate protein kinase C (PKC) and induce 4- to 5-fold increases in the expression of MLC-2 and ANF promoter/luciferase reporter genes with little effect on Rous sarcoma virus/luciferase or minimal prolactin promoter/luciferase genes. To further assess the role of PKC in cardiac gene regulation, PKC expression vectors encoding constitutively activated PKC-alpha or PKC-beta, or a catalytically inactive PKC, were transiently cotransfected with the cardiac promoter/luciferase constructs. Cotransfection of either activated PKC-alpha or PKC-beta cDNA induces the expression of MLC-2 and ANF promoter/luciferase genes and of a reporter gene responsive to the transcription factor AP-1. The Rous sarcoma virus/luciferase and minimal prolactin promoter/luciferase genes are not concomitantly induced by cotransfectin with the PKC genes, indicating specificity of the transcriptional effect. The finding that activated PKC increases cardiac gene transcription suggests that activation of this enzyme may be a proximal signal for coregulation of two cardiac genes, MLC-2 and ANF, during the course of myocardial cell hypertrophy.
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PMID:Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes. 153 37

Gene transfer therapy in vascular surgery is on the horizon and will include the insertion of genes for anti-clotting proteins into the endothelial lining of vascular grafts and for genes controlling the proliferation of smooth muscle cells after endovascular intervention. Here we address the possibility of targeting genes to specific vascular cells using non-infectious methods, DEAE-dextran or lipofectin complexes of reporter genes, to aid transfection of endothelial cells, smooth muscle cells and fibroblasts cultured from human umbilical veins or arteries. For these studies we used the firefly luciferase gene under control of several different promoters including those for the Rous sarcoma virus (RSV) and for tissue plasminogen activator type 1 (PAI-1). DEAE-dextran mediated transfections resulted in low level, transient (2-5 days) expression of RSV-luciferase in all three cell types. Lipofectin mediated transfections resulted in a four-to-five-fold higher expression of RSV-luciferase in endothelial and smooth muscle cells, expression remaining fairly stable for up to 14 days. One particular PAI-1 promoter construct of 800 bp was only half as effective as the RSV promoter in the expression of luciferase from smooth muscle cells, 82 +/- 9 and 35 +/- 11 ng mg-1 respectively (p less than 0.02). In contrast these two promoters resulted in very similar expression of luciferase in endothelial cells, 64 +/- 8 and 67 +/- 10 ng mg-1 respectively. These experiments demonstrate the possibilities of augmenting cultured vascular cells with foreign genes using lipofectin, a cationic lipid, for insertions into endothelial and smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gene transfer into specific vascular cells. 157 52

Biologically active replication-competent (subgroups A, B, and C) and replication-defective Rous sarcoma virus-derived vectors containing the cDNA encoding firefly luciferase as a reporter gene were constructed. In these retroviral vectors, luciferase is expressed from a spliced subgenomic mRNA. A biologically active replication-defective UR2 virus-derived vector expressing the reporter gene as a gag-luciferase fusion protein from an unspliced genomic mRNA was also constructed. The luciferase reporter gene was used because it lacks homology with chicken genomic sequences and because a rapid and sensitive direct enzymatic assay is available to monitor luciferase expression in retrovirus-infected cells. The levels of luciferase expression in luciferase recombinant retrovirus-infected chicken embryo fibroblasts are greater than 10(3) higher than that detected in uninfected cells or in cells infected with retroviral vectors carrying other genes. Endpoint dilution titration experiments demonstrated that one infected cell can be detected in a background of 10(3) uninfected cells. The vectors are stable in tissue culture and high level expression of the unselected luciferase reporter gene is maintained. The vectors were used to express luciferase in chicken embryos, demonstrating the potential utility of luciferase as a reporter in vivo.
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PMID:Avian retroviral expression of luciferase. 166 Jan 98

Overexpression of the p185 product of the c-erb B2/neu gene is correlated with cell transformation and tumorigenesis. To study expression of this gene, a 1548-base pair fragment (-1571 to -24 bp relative to the ATG initiator codon) of the human c-erb B2/neu 5'-noncoding region was isolated, sequenced, and analyzed with respect to basal and inducible activity using the luciferase expression vector pSVOAL delta 5'. This fragment contained an Alu repetitive element which was confirmed in two independent clones. Basal activity of the 1548-base pair fragment was equivalent to the epidermal growth factor receptor promoter region and 32 and 16% as active as the Herpes simplex thymidine kinase and Rous sarcoma virus promoters, respectively. Induction of luciferase activity was observed in response to epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, dibutyryl cAMP, and retinoic acid. Additive and synergistic responses with more than 30-fold increases were observed after treatment with combinations of inducing agents, indicating complex regulation of this gene. These results show that the promoter region of the c-erb B2/neu gene contains sequences that dictate regulatory responses to several environmental signals.
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PMID:Structure and inducible regulation of the human c-erb B2/neu promoter. 196 58

Transgenic chickens were produced by injecting the Day-1 egg with 10(5) infectious particles of a replication-competent virus based on the Schmidt-Ruppin A strain of Rous sarcoma virus. The chickens were resistant to transforming subgroup A virus containing the src gene but not the corresponding subgroup B virus. Transgenic chickens producing bovine growth hormone (bGH) were generated using a modified virus containing the Bryan high titre polymerase gene. The virus was constructed with the bGH gene and the mouse metallothionein promoter in the reverse orientation relative to the viral structural genes. Two male chickens produced serum concentrations of approximately 100 ng bGH/ml; the birds were larger than controls and matured more rapidly. Transgenic mice required for the analysis of skeletal muscle-specific expression in vivo were produced using 5'-flanking regions of the chicken alpha-skeletal actin promoter linked to a luciferase reporter gene to determine the region essential for tissue-specific expression. The defined promoter sequences are to be used in experiments designed to direct expression of growth-promoting genes in skeletal muscle of chickens.
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PMID:Vectors, promoters, and expression of genes in chick embryos. 217 Jun 40

The small marine ostracod crustacean, Vargula hilgendorfii, produces a bright blue luminous secretion which is ejected into seawater. The luminescence is due to a simple enzyme-catalyzed reaction involving only luciferase, luciferin (substrate), and molecular oxygen. Thus, V. hilgendorfii luciferase (VL) should be useful as a reporter enzyme in studies of gene expression in mammalian cells. Expression plasmids consisting of VL cDNA (vl) linked to the promoters simian virus 40 early region, Rous sarcoma virus long terminal repeat, human elongation factor, or mouse granulocyte colony-stimulating factor were introduced into a series of mammalian cell lines. Following transfection, VL activities in cell extracts and culture media were determined by a rapid light emission assay with V. hilgendorfii luciferin. Parallel experiments were carried out with the chloramphenicol acetyltransferase (CAT)-encoding gene. In all cell lines tested, VL was secreted, allowing the reporter activity to be determined directly from a small aliquot of the culture medium. The results indicate that the secreted VL enzyme is superior to CAT, firefly luciferase, and bacterial luciferase as a convenient and versatile indicator of gene expression in mammalian cells.
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PMID:Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. 226 35

The human P450IIE1 gene, coding for an ethanol-inducible nitrosamine-metabolizing P-450, was isolated from a lambda EMBL3 genomic library and completely sequenced. The human gene spanned 11,413 base pairs and contained nine exons and a typical TATA box. Upstream and downstream DNAs of 2788 and 559 base pairs were also sequenced and compared to the rat gene. Significant areas of sequence similarity were observed within 140 base pairs upstream of the transcription start site in the rat and human genes. Human DNA 539 base pairs upstream of the transcription start site was inserted into the expression vector pSVOAL delta 5', and luciferase activity was detected when the constructs were introduced into a rat hepatoma cell line. The activity was over 100-fold lower than that of pRSVL, a Rous sarcoma virus LTR-driven luciferase gene. By use of panels of rodent-human cell hybrids, the gene was mapped to chromosome 10 (CYP2E locus). A full-length cDNA, constructed with the first exon of the genomic clone and a partial cDNA clone, was expressed in COS cells and found to code for N-nitrosodimethylamine demethylase activity.
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PMID:Human ethanol-inducible P450IIE1: complete gene sequence, promoter characterization, chromosome mapping, and cDNA-directed expression. 323 19


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